Method involving submicroliter crystallization and subsequent scale up experiment

ABSTRACT

A method is provided for performing array microcrystallizations to determine suitable crystallization conditions for a molecule, the method comprising: forming an array of microcrystallizations, each microcrystallization comprising a drop comprising a mother liquor solution whose composition varies within the array and a molecule to be crystallized, the drop having a volume of less than 1 microliter; storing the array of microcrystallizations under conditions suitable for molecule crystals to form in the drops in the array; and detecting molecule crystal formation in the drops by taking images of the drops.

CROSS-REFERENCE TO RELATED APLLICATIONS

[0001] This application is a Continuation-in-Part of U.S. applicationSer. No. 09/336,134. filed Jun. 18, 1999, which is incorporated hereinby reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to methods and apparatuses forcrystallizing molecules and, more particularly, to methods andapparatuses for automating the crystallization of molecules,particularly macromolecules such as proteins.

[0004] 2. Description of Related Art

[0005] Fast progress in the area of genomics has provided explosivelygrowing databases of information on genes of human and other organismsby mapping, sequencing and analyzing their genomes. Many genes that maybe critical for identifying people predisposed to certain diseases suchas cancer have been discovered and their biological functions have beenassessed in vitro and/or in vivo. Recently, a new area of genomics,functional genomics, has been developed, which involves a genome wideanalysis of gene function by using information and reagents from thegenomic analysis and expressing the genes in various organisms such asyeast. Functional genomics has generated important information regardingthe expression pattern of genes by using high throughput screeningtechniques such as DNA oligonucleotide chips for specific genes or highdensity microarrays. An understanding of the network of interactionsbetween a protein expressed by a target gene and other macromolecules inthe cell is also being expanded at an unprecedented rate by usingefficient screening methods such as the yeast hybrid systems.

[0006] One of the ultimate goals of these genome projects is thedevelopment of efficacious therapeutics against proteins expressed bydisease genes. Among various methods of drug discovery and development,structure-based drug development has become one of the most importantapproaches, thanks to rapidly advancing computation techniques. It iswell recognized that understanding of the detailed three-dimensionalstructure of a protein not only assists in rational drug design anddevelopment in the laboratory but also provides a well-defined target inhigh throughput drug screening by using computer-aided docking analysis.

[0007] Solving high resolution structures of protein in a highthroughput fashion presents a major bottleneck in such a chain ofgenomics and drug development. High resolution structures of proteinsare solved by X-ray crystallography, and more recently by usingmulti-dimensional NMR spectroscopy on high-field NMR machines forsmaller proteins or peptides.

[0008] Various methods for X-ray crystallography have been developed,including the free interface diffusion method (Salemme, F. R. (1972)Arch. Biochem. Biophys. 151:533-539), vapor diffusion in the hanging orsitting drop method (McPherson, A. (1982) Preparation and Analysis ofProtein Crystals, John Wiley and Son, New York, pp 82-127), and liquiddialysis (Bailey, K. (1940) Nature 145:934-935).

[0009] Presently, the hanging drop method is the most commonly usedmethod for growing macromolecular crystals from solution, especially forprotein crystals. Generally, a droplet containing a protein solution isspotted on a cover slip and suspended in a sealed chamber which containsa reservoir with a higher concentration of precipitating agent. Overtime, the solution in the droplet equilibrates with the reservoir bydiffusing water vapor from the droplet, thereby slowly increasing theconcentration of the protein and precipitating agent within the droplet,which in turn results in precipitation or crystallization of theprotein.

[0010] The process of growing crystals with high diffraction quality istime-consuming and involves trial-and-error experimentations on multiplesolution variables such as pH, temperature, ionic strength, and specificconcentrations of salts, organic additives, and detergents. In addition,the amount of highly purified protein is usually limited,multi-dimensional trials on these solution conditions is unrealistic,labor-intensive and costly.

[0011] A few automated crystallization systems have been developed basedon the hanging drop methods, for example Cox, M. J. and Weber, P. C.(1987) J. Appl. Cryst. 20:366; and Ward, K. B. et al. (1988) J. CrystalGrowth 90:325-339. A need exists for improved automated crystallizationsystems for proteins and other macromolecules.

SUMMARY OF THE INVENTION

[0012] The present invention relates to a method for performing arraymicrocrystallizations to determine suitable crystallization conditionsfor a molecule. The molecule may be a molecule for which an x-raycrystal structure is needed. Determining high-resolution structures ofmolecules by a high-throughput method such as the one of the presentinvention can be used to accelerate drug development. The molecule to becrystalized may also be a molecule for which a crystalline form of themolecule is needed. For example, it may be desirable to create acrystalline form of a molecule or to identify new crystalline forms of amolecule. In some instances, particular crystalline forms of a moleculemay have more bioactive, dissolve faster, decompose less readily, and/orbe easier to purify,

[0013] The molecule is preferably a macromolecule such as a protein butmay also be other types of macromolecules. The molecule preferably has amolecular weight of at least 500 Daltons, more preferably at least 1000Daltons, although smaller molecular weight molecules may also becrystallized.

[0014] In one embodiment, the method comprises: forming an array ofmicrocrystallizations, each microcrystallization including a dropcontaining a molecule to be crystallized and a mother liquor solutionwhose composition varies within the array, the drop having a volume ofless than 1 L; storing the array of microcrystallizations underconditions suitable for molecule crystals to form in the drops in thearray; and detecting molecule crystal formation in the drops.

[0015] In one variation, the method comprises: forming an array ofmicrocrystallizations, each microcrystallization comprising a wellincluding a mother liquor solution whose composition varies within thearray, and drop region including a drop containing the molecule to becrystallized, the drop having a volume of less than 1 L; storing thearray of microcrystallizations under conditions suitable for moleculecrystals to form in the drops in the array; and detecting moleculecrystal formation in the drops.

[0016] In another variation, the method comprises: forming an array ofmicrocrystallizations, each microcrystallization comprising a wellincluding a mother liquor solution whose composition varies within thearray, and a coverslip including a drop containing the molecule to becrystallized, the drop having a volume of less than 1 L; storing thearray of microcrystallizations under conditions suitable for moleculecrystals to form in the drops in the array; and detecting moleculecrystal formation in the drops.

[0017] In yet another variation, the method comprises: forming an arrayof microcrystallizations, each microcrystallization comprising a wellincluding a mother liquor solution whose composition varies within thearray, and sitting drop region including a drop containing the moleculeto be crystallized, the drop having a volume of less than 1 L; storingthe array of microcrystallizations under conditions suitable formolecule crystals to form in the drops in the array; and detectingmolecule crystal formation in the drops.

[0018] According to any of the above methods, the volume of the dropcontaining the molecule to be crystallized is less than about 1 L,preferably less than about 750 nL, more preferably less than about 500nL, and most preferably less than about 250 nL. In one variation, thedrop volume is between 1 nL and 1000 nL, preferably between 1 nL-750 nL,more preferably between 1 nL-500 nL, more preferably between 1 nL-250nL, and most preferably between 10 nL-250 nL.

[0019] The present invention also relates to plates for performing arraymicrocrystallizations to determine suitable crystallization conditionsfor a molecule. According to one embodiment, the plate comprises anarray of at least 36 wells for holding a mother liquor solution, eachwell having a reservoir volume of less than about 500 L, preferably lessthan about 400 L, more preferably less than about 300 L and optionallyless than about 250 L. Ranges of well volumes that may be used include,but are not limited to 25 L-500 L and 25 L-300 L. In one variation, theplate is designed to perform a hanging drop crystallization. In anothervariation, the plate is designed to perform a sitting dropcrystallization and includes a mother liquor well as well as an adjacentsitting drop well.

[0020] The present invention also relates to various apparatuses forforming submicroliter drops used in an array microcrystallization todetermine suitable crystallization conditions for a molecule.

[0021] In one embodiment, the apparatus comprises:

[0022] a platform on which a multiwell plate is positionable;

[0023] a mother liquor drop station capable of removing mother liquorfrom a plurality of wells of the multiwell plate and deliveringsubmicroliter volumes of mother liquor to drop regions on the multiwellplate within a volume range of less than about 25 nL; and

[0024] a molecule drop station capable of delivering submicrolitervolumes of a solution containing a molecule to be crystallized to thedrop regions within a volume range of less than about 25 nL.

[0025] In another embodiment the apparatus is designed for preparingsubmicroliter hanging drops on cover slips used in an arraymicrocrystallization, the apparatus comprising:

[0026] a platform on which a multiwell plate is positionable;

[0027] a cover slip station on which a plurality of coverslips arepositionable;

[0028] a mother liquor drop station capable of removing mother liquorfrom a plurality of wells of the multiwell plate and deliveringsubmicroliter volumes of mother liquor to the plurality of coverslipswithin a volume range of less than about 25 nL; and

[0029] a molecule drop station capable of delivering submicrolitervolumes of a solution containing a molecule to be crystallized to theplurality of coverslips within a volume range of less than about 25 nL.

[0030] In yet another embodiment the apparatus is designed for preparingsubmicroliter sitting drops used in an array microcrystallization, theapparatus comprising:

[0031] a platform on which a multiwell plate is positionable;

[0032] a mother liquor drop station capable of removing mother liquorfrom a plurality of wells of the multiwell plate and deliveringsubmicroliter volumes of mother liquor to drop regions on the multiwellplate within a volume range of less than about 25 nL; and

[0033] a molecule drop station capable of delivering submicrolitervolumes of a solution containing a molecule to be crystallized to thedrop regions within a volume range of less than about 25 nL.

[0034] According to any of the above embodiments, the mother liquor dropstation and the molecule drop station are each capable of deliveringsubmicroliter volumes within a volume range of less than about 20 nL.more preferably less than 15 nL, and most preferably less than 10 nL.

[0035] Also according to any of the above embodiments. a sensor may beincluded in the apparatus for preparing submicroliter drops which isdetects whether mother liquor drops and/or molecule drops have beenformed.

[0036] The mother liquor drop station and the molecule drop station arepreferably each independently capable of delivering submicrolitervolumes to at least four coverslips at a time, more preferably at leasteight coverslips at a time.

[0037] The present invention also relates to methods for formingsubmicroliter drops for use in an array microcrystallization todetermine suitable crystallization conditions for a molecule. Accordingto one embodiment, the method includes: removing mother liquor from aplurality of wells of a multiwell plate; delivering submicrolitervolumes of the mother liquor to drop regions of the multiwell platewithin a volume range of less than about 25 nL; and deliveringsubmicroliter volumes of a solution containing a molecule to becrystallized to the drop regions of the multiwell plate within a volumerange of less than about 25 nL; wherein a total volume of thesubmicroliter volumes delivered to each drop region is less than 1 L.

[0038] According to another embodiment, the method is for a hanging dropcrystallization and includes: taking a plurality of coverslips; removingmother liquor from a plurality of wells of a multiwell plate; deliveringsubmicroliter volumes of the mother liquor to the plurality ofcoverslips within a volume range of less than about 25 nL; anddelivering submicroliter volumes of a solution containing a molecule tobe crystallized to the plurality of coverslips within a volume range ofless than about 25 nL; wherein a total volume of the submicrolitervolumes delivered to each coverslip is less than 1 L.

[0039] According to another embodiment, the method is for a sitting dropcrystallization and includes: removing mother liquor from a plurality ofwells of a multiwell plate; delivering submicroliter volumes of themother liquor to sitting drop regions of the multiwell plate within avolume range of less than about 25 nL; and delivering submicrolitervolumes of a solution containing a molecule to be crystallized to thesitting drop regions within a volume range of less than about 25 nL;wherein a total volume of the submicroliter volumes delivered to eachsitting drop region is less than 1 L.

[0040] According to any of the above method embodiments, the totalvolume of the submicroliter volumes delivered is preferably less thanabout 750 nL, more preferably less than about 500 nL, and mostpreferably less than about 50 nL. It is noted that the drop volumes maybe as small as 380 pL. The volumes delivered preferably range between 1nL-750 nL, more preferably between 1 nL-500 nL, more preferably between1 nL-250 nL, and most preferably between 10 nL-250 nL.

[0041] According to any of the above apparatus and method embodiments,the precision of the volumes delivered is preferably less than about 25nL, more preferably less than 20 nL, more preferably less than 15 nL,and most preferably less than 10 nL. The precision of the volumesdelivered may also be between 380 pL and 25 nL, more preferably between380 pL and 20 nL. more preferably between 380 pL and 15 nL, and mostpreferably between 380 pL and 10 nL.

BRIEF DESCRIPTION OF THE FIGURES

[0042]FIG. 1 is a block diagram of a crystallization system according tothe present invention.

[0043]FIG. 2 illustrates a method for using the crystallization systemof FIG. 1 to perform a protein crystallization trial.

[0044]FIG. 3A illustrates a top view of a multiwell plate which may beused to perform a hanging drop array microcrystallization.

[0045]FIG. 3B is a sideview of the multiwell plate illustrated in FIG.3A.

[0046]FIG. 3C illustrates a top view of a multiwell plate which may beused to perform a sitting drop array microcrystallization.

[0047]FIG. 3D is a sideview of the multiwell plate illustrated in FIG.3C.

[0048]FIG. 3E is a cross section of a multiwell plate with a platecover.

[0049] FIGS. 4A-4J illustrate the various stations which can be includedin a mother liquor delivery system.

[0050]FIG. 4A is a sideview of a plate loading station looking across aplate track positioned adjacent to the plate loading station.

[0051]FIG. 4B is a sideview of a plate loading station looking along thelongitudinal axis of the plate track.

[0052]FIG. 4C is a sideview of a bar code reading station for reading abar code included on a multiwell plate.

[0053]FIG. 4D is a sideview of a sealing medium station for applying asealing medium to an upper edge of wells defined in a multiwell plate.

[0054]FIG. 4E is a sideview of a plate cover removal station forremoving a plate cover from a multiwell plate.

[0055]FIG. 4F is a topview of a mother liquor delivery station.

[0056]FIG. 4G is a topview of a delivery block for delivery of a motherliquor into multiwell plates.

[0057]FIG. 4H is a cross section of a delivery block for delivery of amother liquor into a multiwell plates.

[0058]FIG. 4I is a sideview of a mother liquor source storage bank.

[0059]FIG. 4J is a sideview of a syringe pump for delivering a motherliquor from a mother liquor source to a fluid injector.

[0060] FIGS. 5A-5E illustrate the various stations which can be includedin a drop formation system.

[0061]FIG. 5A is a top view of a drop formation station.

[0062]FIG. 5B is a sideview of the drop formation station.

[0063]FIG. 5C is a sideview of a pipette holder.

[0064]FIG. 5D is a sideview of a well cover holder.

[0065]FIG. 5E is a sideview of a well cover magazine for storing wellcovers to be positioned over the wells in a multiwell plate.

[0066] FIGS. 6A-6I illustrate operation of the drop formation station.

[0067]FIG. 6A illustrates a drop formation station in the rest position.

[0068]FIG. 6B illustrate the drop formation station with a multiwellplate has been moved into position for drop formation and a pipetteholder is moved into position over the wash basin.

[0069]FIG. 6C illustrates the pipette holder moved into position over acolumn of wells in the multiwell plate.

[0070]FIG. 6D illustrates the pipette holder moved into position overthe well cover holder.

[0071]FIG. 6E illustrates the pipette holder returned to its restposition and a protein delivery pipette moved into position over a wellcover.

[0072]FIG. 6F illustrates the protein delivery pipette moved into itsrest position and the cover holder inverted and moved into position overthe column of wells on the multiwell plate.

[0073]FIG. 6G illustrates hanging drops suspended from well covers overthe wells of a plate.

[0074]FIG. 6H illustrates the cover holder moved into position over awell cover storage component.

[0075]FIG. 6I illustrates the cover holder returned to its restposition.

[0076] FIGS. 7A-7G illustrate operation of the drop formation station toform sitting drops.

[0077]FIG. 7A illustrates a drop formation station in the rest position.

[0078]FIG. 7B illustrate the drop formation station with a multiwellplate adapted to perform a sitting drop array microcrystallization inposition for drop formation and a pipette holder moved into positionover the wash basin.

[0079]FIG. 7C illustrates the pipette holder moved into position over acolumn of wells in the multiwell plate.

[0080]FIG. 7D illustrates pipettes in the pipette holder aligned withthe well regions of wells in a column of the plate.

[0081]FIG. 7E illustrates pipettes in the pipette holder aligned withthe sitting drop regions of wells in a column of the plate.

[0082]FIG. 7F illustrates the protein delivery pipette moved intoposition over the sitting drop region of a well in the column of wells.

[0083]FIG. 7G illustrates a sitting drop formed in the sitting dropregion of a well.

[0084]FIG. 8A is a sideview of a plate track with a pin extending abovethe plate track from a pin carriage positioned beneath the plate track.

[0085]FIG. 8B is a sideview of a plate track with a pin of FIG. 8Awithdrawn beneath the plate track.

[0086]FIG. 8C is a sideview of a transport assembly having a pluralityof pin carriages.

[0087]FIG. 9 illustrates the composition of 480 mother liquor solutionsfor a preferred coarse screen.

[0088] FIGS. 10A-10D illustrate formation of crystals in different dropssized from 40 nL to 1000 nL.

[0089]FIG. 11 lists the mother liquor compositions for 24 mother liquorsused in the fine screen stage of a crystallization trial.

[0090]FIG. 12A illustrates an optical system for screeningcrystallization experiments for crystals.

[0091]FIG. 12B illustrates an optical system for screening small dropvolume crystallization experiments for crystals.

DETAILED DESCRIPTION

[0092] The present invention relates to a method for performing arraymicrocrystallizations to determine suitable crystallization conditionsfor a molecule. The molecule is preferably a macromolecule, such as aprotein. Other types of molecules and macromolecules may also becrystallized according to the present invention. The molecule preferablyhas a molecular weight of at least 500 Daltons, more preferably at least1000 Daltons, although it is noted that the invention can be applied tomolecules with lower molecular weights.

[0093] The method involves forming an array of microcrystallizationswhere each microcrystallization includes a drop containing a molecule tobe crystallized and a mother liquor solution whose composition varieswithin the array, the drop having a volume of less than 1 L. The arrayof microcrystallizations are stored under conditions suitable formolecule crystals to form in the drops in the array. Molecule crystalformation is then detected in the drops. As will be described herein,this method can be employed in any crystallization method involvingdrops, including, but not limited to hanging drop crystallizationmethods and sitting drop crystallization methods. Example sitting dropcrystallization methods are provided in U.S. Pat. No. 5,096,676(McPherson et al.) and U.S. Pat. No. 5,419,278 (Carter).

[0094] An important feature of the present invention is the utilizationof small drop volumes. For example, the volume of the drop containingthe molecule to be crystallized is less than about 1 L, preferably lessthan about 750 nL. more preferably less than about 500 nL, and mostpreferably less than about 250 nL. In one variation, the drop volume isbetween 1 nL and 1000 nL, preferably between 1 nL-750 nL, morepreferably between 1 nL -500 nL, more preferably between 1 nL-250 nL,and most preferably between 10 nL-250 nL.

[0095] Applicants believe that the rate of crystallization is dependenton the drop volume where crystals form faster when smaller drop volumesare used. As a result, crystals can be formed more rapidly by using thesmaller drop volumes used in the present invention. This significantlyincreases the through-put rate of the method for determiningcrystallization conditions.

[0096] Without being bound by theory, it is believed that smaller dropswill equilibrate faster than larger drops and that this causes crystalsto form more rapidly. The rate of equilibration is believed to berelated to a relationship between the rate of drop evaporation and dropvolume. Meanwhile, the rate of drop evaporation is dependent on dropsurface area. The surface area of a drop does not decrease linearly withthe drop's volume. As a result, a larger drop having twenty times thevolume of a smaller drop (e.g., 1 L vs. 50 nL) will have significantlyless than twenty times the surface area of the smaller drop. By reducingdrop volume, one is able to improve the relationship between the rate ofdrop evaporation (surface area dependent) and drop volume, therebyaccelerating equilibration and crystal formation.

[0097] A further advantage of the present invention is that smaller dropvolumes allow less molecule to be used to perform each crystallizationtrial.

[0098] As a result, a greater number of crystallization trials can beperformed using the same amount of molecule. This is of greatsignificance when it is difficult to obtain the molecule to becrystallized and when a large number of crystallization trials areneeded in order to successfully crystallize the molecule.

[0099] It is frequently difficult to produce and purify the moleculebeing crystallized. In the case of protein crystallization, it canrequire one to two weeks of lab work to produce and purify enoughprotein to perform 48 crystallization trials using drops greater than 1L in size. By reducing the drop volume and hence the amount of moleculeused per crystallization trial, it becomes feasible to significantlyincrease the number of crystallization trials that can be performed. Asa result, it becomes feasible to take a more combinatorial, shotgunapproach to molecule crystallization trials since the pressure toconserve molecule usage is reduced. By contrast, prior to the presentinvention's utilization of sub microliter drop volumes, a need existedto minimize the number of trials that were performed at one time due toa shortage of available molecule.

[0100] By reducing the drop volume, the number of microcrystallizationsthat can be performed in the array is increased. The number ofmicrocrystallizations in the array is typically greater than 48,preferably greater than 96, more preferably greater than 144, mostpreferably greater than 192. It is noted that the number ofmicrocrystallizations in the array can also exceed 288 or 384. Forexample, an apparatus for preparing arrays which include 480microcrystallizations is described herein.

[0101] Increasing the number of microcrystallizations that can beperformed in the array also allows a greater number of different stocksolutions to be used to form the mother liquor solutions used in thearray. For example, forming the array of microcrystallizations caninclude using greater than 48 stock solutions to form the mother liquorsolutions used in the array. Optionally, greater than 96, morepreferably greater than 144, most preferably greater than 192 differentstock solutions may be used. It is noted that the number of stocksolutions can also exceed 288 or 384. For example, an apparatusdescribed herein uses 480 different stock solutions.

[0102] Smaller volumes of mother liquor may also be used in the wells.The volume of mother liquor used in the wells is preferably less thanabout 500 L, preferably less than about 400 L, more preferably less thanabout 300 L and optionally less than about 250 L. Ranges of motherliquor volumes that may be used include, but are not limited to 25 L-500L and 25 L-300 L. In this regard, forming the array ofmicrocrystallizations may include forming the microcrystallizations in aplate including a plurality of wells each having a volume less thanabout 500 L, preferably less than about 400 L, more preferably less thanabout 300 L.

[0103] The use of small volumes of mother liquor allows the wells inmultiwell plates to be made smaller, thereby allowing more wells to bepositioned on a multiwell plate per unit area. For example, the 48 wellplates having a well volume less than about 500 L has approximately thesame footprint as 24 well plates typically used to perform proteincrystallization. Further reduction of the mother liquor volumes may beemployed in order to further reduce plate sizes.

[0104] By utilizing small drop volumes, a significantly greater numberof crystallization trials can be performed using the same amount ofmolecule. As a result, it is feasible to perform a greater number ofcrystallization trials, which in turn allows the mother liquor solutionto be more widely varied in its composition. This allows the motherliquor solution to be formed of 1, 2, 3, 4, 5, 6 or more componentswhich are varied within the array.

[0105] Also according to this method, the array of microcrystallizationsis formed of one or more multiwell plates. Each plate preferably has atleast 24 wells, more preferably at least 36 wells, and most preferablyat least 48 wells. By utilizing less mother liquor, smaller wells can beused which allows the same size plate to contain more wells.

[0106] Also according to this method, detecting crystal formation caninclude characterizing the crystal formed (needle, cube, etc.), the sizeof the crystal, and the quality of the crystal's structure.Characterization of the crustal can be performed manually, or by takingimages of the drops and analyzing those images for the structure ofcrystals contained within those drops.

[0107] As noted elsewhere, an objective of the present invention is toprovide a high throughput methodology for testing crystallizationconditions. By reducing crystallization volumes, the present inventionallows one to perform many more crystallization experiments using thesame amount of protein. However, when one performs many morecrystallization experiments, it then becomes necessary to screen thesemany more crystallization experiments for crystals.

[0108]FIG. 12A illustrates an optical system 210 for screeningcrystallization experiments for crystals. As illustrated, the opticalsystem 210 has an image plane 212. Objects 214 which are transected bythe image plane 212 are in focus. Objects outside the image plane 212are not in focus. Depending on the depth of field of the optics used,objects outside the image plane can be seen, but with decreasedresolution. Ultimately, the depth of field that can be imaged isdependent on the nuerical aperture of the optical system.

[0109] The positioning of the image plane 212 is dependent upon thefocal length of the optics used in the optical system and thepositioning of the optical system 210 relative to the object 214 to beimaged. Focusing the optical system 210 causes the image plane 212 tomove vertically toward or away from the optical system 210.

[0110]FIG. 12A illustrates a crystallization experiment where thecrystallization volume employed (in this case a drop) is larger thanthose used in the present invention. As illustrated, when larger dropvolumes are employed, the vertical thickness 216 of the drop 218 is suchthat crystals 220 can be present in the drop and outside of the imageplane 212 of optical system 210. As a result, it is necessary to adjustthe focus of the optical system 210. This causes the image plane 212 tomove vertically across the vertical thickness 216 of the drop 218 sothat the entire drop can be screened for crystals.

[0111] An advantageous feature of performing crystallizations usingsmall drop volumes according to the present invention is that the needto focus the optical system in order to screen for crystals within thedrop is eliminated. As illustrated in FIG. 12B, when one images a drophaving a small drop volume, the vertical thickness 216 of the drop 218is sufficiently small that a crystal, if present, will transect thefocal plane 212 of the optical system if the focal plane is positionedwithin the drop, preferably around the middle of the drop. This is shownexperimentally herein with regard to FIGS. 10A-10D. As a result, it isunnecessary to adjust the focus the optical system in order scan a dropfor crystals. As a result, a single image of a drop can be taken inorder to evaluate whether crystals are present in the drop.

[0112] As noted above, by reducing drop volumes, the present inventionallows one to perform many more crystallization experiments using thesame amount of protein. At the same time, the reduced drop volumes ofthe present invention also allows one to screen crystallizationexperiments for crystals more rapidly by eliminating the need to adjustthe focus of an optical system used to image the crystallizationexperiments.

[0113] The method can also include identifying the compositions of thosemother liquor solutions in which crystals were detected and performingadditional crystallization trials where the formulation of the motherliquor solutions in which crystals were detected is further varied.

[0114]FIG. 1 illustrates a crystallization system 10 for performing acrystallization trial. The crystallization system 10 can be divided intovarious stations 12 described below. During operation of thecrystallization system 10, multiwell plates are positioned on a platetrack 14. A transport assembly (not shown) moves the multiwell platesalong the plate track 14 to the various stations 12.

[0115] The crystallization system 10 also includes a processing unit 16in electrical communication with the various stations 12. Suitableprocessing units 16 for use with the crystallization system 10 include,but are not limited to, PCs and computer workstations. The processingunit 16 includes process control logic for controlling the operation ofeach station and the transport assembly. An operator can use one or moreuser interfaces to interact with, disengage and/or to alter the processcontrol logic. Suitable user interfaces include, but are not limited tocomputer monitors, keyboards, mouses. and trackballs.

[0116] During operation of the crystallization system 10, the transportassembly moves a multiwell plate past the stations 12 which each performa particular function. For instance, the crystallization system 10includes a plate loading station 18 where multiwell plates aresequentially loaded onto the plate track 14. The crystallization system10 also includes a bar code reading station 20 where a bar code on themultiwell plates can be read. The crystallization system 10 furtherincludes a sealing medium station 22. The sealing medium station 22 canbe used to apply a sealing medium to the multiwell plates. Specifically,the sealing medium can be applied to the upper edge of liquid receivingwells defined in each multiwell plate. The sealing medium serves to forma seal between the upper edges of each well and a well cover, commonlyreferred to as a coverslip, which is positioned over each well at alater station of the crystallization system 10. The crystallizationsystem 10 also includes a plate cover removal station 24 where platecovers 44 are delivered to or removed from the multiwell plates.

[0117] The crystallization system 10 also includes a mother liquordelivery station 26 where mother liquors are delivered into the wellsdefined in the multiwell plates. Different mother liquors can bedelivered into different wells or the same mother liquor can bedelivered into more than one well. Further. mother liquor can bedelivered into a portion of the wells on a single multiwell plate so theremaining wells are empty.

[0118] The crystallization system 10 also includes a drop formationstation 28 where mother liquors from the various wells are used to formone or more drops on a plurality of coverslips that will be placed overthe wells. The drop formation station 28 also adds a solution containingthe molecule to be crystallized to the coverslips. Once drops containingmother liquor and the molecule to be crystallized are formed on thecoverslips, the coverslips are positioned over each well such that theone or more drops hang from the coverslip into the well. These drops arecalled hanging drops.

[0119] It is noted that the drop formation station can be readilyadapted to form sitting drops in a sitting drop regions of a multiwellplate by delivering mother liquors from the various wells and thesolution containing the molecule to be crystallized to the sitting dropregions.

[0120] The crystallization system 10 also includes a plate coverdelivery station 29 where plate covers 44 can be positioned on eachmultiwell plate. The multiwell plate can then by transported to a plateunloading station 30 where the multiwell plates can be removed from theplate track 14 and stored.

[0121] Although the crystallization system 10 illustrated in FIG. 1 hasthe various stations 12 positioned around a single plate track 14, it isnoted that the various stations 12 can be divided into one or moresub-systems, each optionally having its own track. It is further notedthat many of the stations 12 may optionally be included or excluded fromthe crystallization system 10. Further, the stations 12 can bepositioned in a sequence other than the sequence illustrated in FIG. 1.For instance, the plate cover removal station 24 can be positionedbefore the bar code reading station 20. Additionally, several of thedescribed functions can be carried out at a single station. Forinstance, a plate cover delivery station 29 can be formed integrallywith the drop formation station 28 or the plate unloading station 30.

[0122] The above stations 12 can be included in a single system or caneach be included in different independent sub-systems. For instance, thetray loading station, bar code reading station 20, sealing mediumstation 22 and mother liquor delivery station 26 can be included in asingle mother liquor delivery system 31 while the drop formation station28 and the plate unloading station 30 can be included in an independentdrop formation system 32. Additionally, the functions associated with aparticular station need not be carried out during operation of thecrystallization system 10. For instance, the mother liquors can bedelivered into the wells of a multiwell plate by an external apparatusbefore the multiwell plate enters the crystallization system 10. In suchan instance, when a multiwell plate already containing mother liquorreaches the mother liquor delivery station 26, the mother liquordelivery station 26 can be operated to not deliver mother liquors intothe wells.

[0123] After a microcrystallization array has been prepared byprocessing a multiwell plate through a crystallization system 10 such asthe one illustrated in FIG. 1. drops in the microcrystallization arraycan be observed for the formation of crystals. When crystals are formedin a drop of a particular well, the quality of crystals within the dropcan be graded for various characteristics such as shape, size or timefor crystal formation. When the mother liquors used in each well aredifferent, the crystal grades can be compared to determine which motherliquor was associated with the most desirable crystals. Accordingly,each well serves as a different crystallization experiment whichproduces results which can be compared with the results of othercrystallization experiments.

[0124] A crystallization trial includes comparing the results of severalcrystallization experiments in order to optimize the composition of themother liquor used for crystallization of a particular molecule. FIG. 2illustrates a technique for performing a crystallization trial. A coarsescreen is performed at an initial stage of the trial. In FIG. 2, thecrystallization experiments associated with the coarse screen areillustrated as a plurality of boxes set out in three different arrayswhich are labeled CS₁, CS_(2 and CS) ₃.

[0125] In each array two variables of the mother liquor composition areincrementally varied as shown along the x and y axis associated witheach array. For instance, in CS₁ several crystallization experiments areperformed where the pH is varied from 2-8 in intervals of 2 and the %(NH₄)₂SO₄ is varied from 20-80 in intervals of 20.

[0126] The crystallization experiments in the coarse screen are analyzedto select one or more crystallization experiments which yield the bestcrystals or, if no crystals form, the best crystal-like precipitate. Acoarse screen experiment selected as producing a promising crystal orcrystal-like precipitate is illustrated as a black box in FIG. 2. Finescreens are then performed for the crystallization experiments selectedthrough the course screen.

[0127] A fine screen crystallization experiment is performed bydesigning a crystallization array based on the mother liquor compositionused in a crystallization experiment selected through the course screen,indicated in FIG. 2 as the array labeled FS₁. The compositions of themother liquors used in the fine screen crystallization array areselected by making small variations in the composition of the motherliquor used in the selected experiment from the course screen. Forexample, if the mother liquors used in the course screen had a pHbetween 2-8 and the mother liquor in the selected crystallization had apH of 4.0, the mother liquors used in the fine screen experiments mighthave a pH between 3.4 to 4.6. Further, by focusing the array aroundmother liquors having a pH of about 4, one can reduce the incrementalchange in the value in the fine screen FS₁ For instance, the incrementalchange in the pH during the coarse screen CS₂ shown in FIG. 2 is 2.0while the incremental change of the pH during the fine screen also shownin FIG. 2 is 0.4.

[0128] Crystals formed in each crystallization experiment in the finescreen are analyzed in order to select the one or more crystallizationexperiments yielding the best crystals or crystal-like precipitate. Acrystallization experiment selected during the fine screen experiment isillustrated in FIG. 2 as a box having an X. If the crystals formedduring the fine screen are of a sufficiently high quality, one mightisolate the crystals formed in the experiment and perform x-raydiffraction on the isolated crystals to resolve the molecule's crystalstructure. Alternatively, one might use the mother liquor used in theselected fine screen experiment in order to grow additional crystals.However, if the crystals formed during the fine screen are not of asufficiently high quality, the mother liquor can be further optimized bytaking the mother liquor used in the selected fine screen experiment asthe starting point for an additional fine screen. FIG. 2 illustrates asecond array of fine screen crystallization experiments labeled FS₂. Itis noted that this iterative process of selecting a fine screenexperiment and performing a finer screen array based on a selectedexperiment can be repeated until a suitable mother liquor is identifiedfor use in preparing crystals.

[0129] The microcrystallization methods and apparatuses of the presentinvention may be used to perform the course screen array experimentsdescribed in regard to FIG. 2 in order to analyze a larger set of motherliquors than had previously been feasible with drop sizes larger than 1microliter. It is noted that the fine screen array experiments may alsobe performed using the microcrystallization methods and apparatuses ofthe present invention or may be performed where drop sizes are largerthan 1 microliter.

[0130]FIG. 3A illustrates a top view of a multiwell plate 34 which maybe used with the methods and apparatuses of the present invention toperform a hanging drop array microcrystallization. As illustrated, themultiwell plate 34 includes a support structure 36 defining wells 38arranged in 6 columns and 8 rows. Although FIG. 3A illustrates amultiwell plate 34 with a total of 48 wells 38, the multiwell plate 34can include a different number of wells 38.

[0131]FIG. 3B provides a sideview of the multiwell plate 34 illustratedin FIG. 3A. Each well 38 includes an upper edge 40 extending above thesupport structure 36. The upper edge 40 is preferably wide enough that alayer of a sealing medium, such as grease, can be applied to the upperedge 40. The support structure 36 preferably has a geometry which allowsmultiwell plates 34 to be stacked on top of one another without onemultiwell plate 34 interfering with the well contents of an adjacentmultiwell plate 34.

[0132]FIG. 3C illustrates a top view of a multiwell plate 34 which maybe used with the methods and apparatuses of the present invention toperform a sitting drop array microcrystallization. As illustrated, themultiwell plate 34 includes a support structure 36 defining 48 wells 38arranged in 6 columns and 8 rows. Although a multiwell plate 34 with 48wells 38 is illustrated the multiwell plate 34 can include a differentnumber of wells 38. A well region 41 is adjacent to a sitting dropregion 42. Although the sitting drop region 42 is illustrated as beingcentrally positioned within the well 38, the sitting drop region 42 canbe positioned to one side of the well 38.

[0133]FIG. 3D provides a sideview of the multiwell plate 34 illustratedin FIG. 3C. The sitting drop region 42 extends upward from the bottom ofthe well 38. As illustrated by the cut-away, the sitting drop region 42can include a recess where a sitting drop can be formed. Each well 38includes an upper edge 40 extending above the support structure 36. Theupper edge 40 is preferably wide enough that a layer of a sealingmedium, such as grease, can be applied to the upper edge 40. The supportstructure 36 preferably has a geometry which allows multiwell plates 34to be stacked on top of one another without one multiwell plate 34interfering with the well 38 contents of an adjacent multiwell plate 34.

[0134] A plate cover 44 can be positioned over each multiwell plate 34as illustrated in FIG. 3E. The plate cover 44 can be designed so thecover rests on the upper edges 40 of the wells 38. As illustrated inFIG. 3B an insert 46 can be positioned between the plate cover 44 andthe multiwell plate 34 so the insert 46 rests on the upper edges 40 ofthe wells 38. The insert 46 can be removable from the plate cover 44 orcan be permanently attached to the plate cover 44. The insert 46 can beformed from a flexible material so the insert 46 provides a seal betweenthe insert 46 and the upper edges 40 of the wells 38 in order to reduceevaporation from the wells 38. Suitable materials for the insert 46include, but are not limited to, soft rubbers and other gasket material.

[0135] As illustrated in FIG. 3A, the multiwell plate 34 can include abar code 48 formed on the support structure 36. The multiwell plate 34can also include a surface 50 sized to receive a bar code sticker.Alternatively, a bar code can be formed on a plate cover 44 or the platecover 44 can include a surface sized to receive a bar code sticker. Whenthe multiwell plate 34 or plate cover 44 includes a surface forreceiving a bar code sticker, the bar code sticker is preferablyremovable from the multiwell plate 34 so different bar codes can befixed to a single multiwell plate 34. As will be discussed later, thesebar codes can be used to identify the multiwell plate 34 and/or thecontents of the multiwell plate 34 to the system control logic.

[0136] FIGS. 4A-4J illustrate embodiments of the various stations 12 ofthe mother liquor delivery system 31 illustrated in FIG. 1. FIGS. 4A and4B illustrate a plate loading station 18 for sequentially loadingmultiwell plates 34 onto a plate track 14. FIG. 4A is a sideview of theplate loading station 18 looking across the plate track 14 and FIG. 4Bis a sideview of the plate loading station 18 looking down thelongitudinal axis of the plate track 14. The plate loading station 18includes a tower 52 positioned over the plate track 14 so the platetrack 14 extends outward from the base of the tower 52. The tower 52includes a chute 54 sized to receive a stack of multiwell plates 34arranged one on top of another.

[0137] The plate loading station 18 also includes plate loweringmechanics (not shown) which can be engaged to lower a multiwell plate 34at the bottom of the stack onto the plate track 14. The action ofgravity moves a new multiwell plate 34 into the position of themultiwell plate 34 lowered onto the plate track 14. The clearancebetween the plate tower 52 and the plate track 14 is enough for theplate transport assembly to transport the multiwell plate 34 loweredonto the plate track 14 out from under the tower 52 as illustrated inFIG. 4A. Once the multiwell plate 34 has been transported from beneaththe tower 52, plate lowering mechanics can be re-engaged so a newmultiwell plate 34 at the bottom of the chute 54 is also loaded onto theplate track 14. Because the plate loading station 18 can hold severalmultiwell plates 34 and sequentially position each multiwell plate 34 onthe plate track 14, the mother liquor delivery system 31 can processmany multiwell plates 34 without an operator manually positioning eachmultiwell plate 34 on the plate track 14.

[0138] The plate loading station 18 can be easily adapted into a plate34 unloading station by operating the plate lowering mechanics inreverse. This reverse operation causes a multiwell plate 34 locatedbeneath the tower 52 to be raised from the plate track 14 and added tothe stack of multiwell plates 34 stored within the chute 54.

[0139]FIG. 4C illustrates a bar code reading station 20. A bar codereader 56 is positioned alongside the plate track 14. The bar codereader 56 is directed toward the plate track 14 at an angle whichpermits the bar code reader 56 to read a bar code 48 on a multiwellplate 34 on the plate track 14. As described above, these bar codes canformed on the multiwell plate 34 or can be included on a bar codesticker to be placed on the multiwell plates 34. The bar code reader 56is monitored by the system control logic which associates each bar codewith a particular multiwell plate 34 and/or with particularcharacteristics of a multiwell plate 34. Suitable characteristicsinclude, but are not limited to, the number of wells 38 in the multiwellplate 34, the volume of the wells 38 in the multiwell plate 34, whetherthe multiwell plate 34 includes a plate cover 44, etc.

[0140] The characteristics of a multiwell plate 34 can beadministratively entered in order to indicate information about themultiwell plate 34 to the system control logic. For instance, a user canenter characteristics such as identifying particular mother liquids tobe delivered into particular wells 38 on a multiwell plate 34. Further,if an operator uses an external method to deliver mother liquids intothe wells 38 of the multiwell plate 34, the user can indicate this tothe system control logic. Because various mother liquids are alreadypresent in the wells 38 of the multiwell plate 34, the system controllogic can override mother liquid delivery station in order to avoiddelivering additional mother liquids to the wells 38 of the multiwellplate 34.

[0141] As a multiwell plate 34 moves through the mother liquor deliverysystem 31, the drop formation system 32 and/or through thecrystallization system 10, the characteristics associated with themultiwell plate 34 can optionally be modified by the system controllogic in order to reflect the changing status of the multiwell plate 34.For example, the system control logic can note when mother liquor hasbeen added, or when drops have been formed.

[0142]FIG. 4D illustrates a sealing medium station 22. The sealingmedium station 22 includes a sealing member 60 suspended over the platetrack 14 at a height which permits multiwell plates 34 to be moved underthe sealing member 60. The sealing member 60 includes a sealing surface62 with a plurality of sealing medium injectors 64 arranged so eachsealing medium injector can be concurrently aligned with a well 38 ineach multiwell plate 34. The sealing medium injectors 64 are inhydraulic communication with a sealing medium source 66. Accordingly, asealing medium can be delivered from the sealing medium source 66 to theportion of the sealing surface 62 adjacent to the sealing mediuminjectors 64.

[0143] The sealing member 60 can be coupled with actuators for movingthe sealing member 60 relative to the wells 38 on a multiwell plate 34.The sealing member 60 can be moved vertically over a multiwell plate 34as illustrated by the arrows labeled A. The sealing member 60 can alsobe translated laterally relative to a multiwell plate 34 as illustratedby the arrows labeled B. Suitable actuators include, but are not limitedto, pneumatic pistons, hydraulic pistons and electrically driven motors.

[0144] In operation of the sealing medium station 22, the platetransport assembly transports a multiwell plate 34 into a position wherethe wells 38 in the multiwell plate 34 are positioned beneath thesealing medium injectors 64. The sealing member 60 is lowered until thesealing surface 62 is in contact with the upper edge 40 of the wells 38on the multiwell plate 34. Because the wells 38 of the multiwell plate34 are aligned with the sealing medium injectors 64 before the downwardmotion of the sealing member 60, the upper edge 40 of each well 38encircles a sealing medium injector. Once, the sealing surface 62 is incontact with the upper edges 40 of the wells 38, the sealing member 60is laterally translated. The lateral translation causes the sealingmember 60 to follow a circular path along an edge of the well 38,although other paths may also be used depending on the geometry of thewells 38. This lateral translation transfers the sealing mediumdelivered to the sealing surface 62 to the upper edge 40 of each well38.

[0145] The amount of sealing medium transferred to the upper edge 40 ofeach well 38 depends on the amount of sealing medium present on thesealing surface 62 adjacent to the sealing medium injectors 64. Theamount of sealing medium delivered to the upper edges 40 of the wells 38should be sufficient to create a substantially airtight seal between acoverslip and the upper edge 40 of the well 38. Suitable sealing mediumsinclude, but are not limited to, grease and vasaline.

[0146] It is noted in regard to the sealing medium station 22 that thestation may be readily adapted for use with hanging drop arraycrystallizations as well as with sitting drop array crystallizations. Inregard to each type of drop array crystallization, an airtight sealshould be formed between the edges of a well 38 and a coversilp or otherform of covering member which is placed over the well 38.

[0147]FIG. 4E is a sideview of a plate cover 44 removal station 24positioned adjacent a plate track 14. The plate cover 44 removal station24 includes a carriage 68 configured to move vertically as illustratedby the arrow labeled A and laterally as illustrated by the arrow labeledB. A plurality of vacuum fittings 70 are coupled with the carriage 68and are in pneumatic communication with a releasable vacuum source.Suitable vacuum fitting include, but are not limited to, rubber fittingshaving a cup shape and including a vacuum port in pneumaticcommunication with a vacuum source.

[0148] During operation of the plate cover removal station 24, the platetransport assembly moves a multiwell plate 34 into position next to theplate cover removal station 24. If the multiwell plate 34 has a platecover 44, the carriage 68 is moved laterally until each of the vacuumfittings 70 are positioned over the multiwell plate 34. The carriage 68is lowered until at least a portion of the vacuum fittings 70 are incontact with the plate cover 44. The vacuum source is activated in orderto immobilize the plate cover 44 relative to the carriage 68. Thecarriage 68 is then raised to its original height. The vertical motionof the carriage 68 lifts the plate cover 44 from the multiwell plate 34.The carriage 68 is then moved laterally until the carriage 68 ispositioned over a plate cover storage component 72. The carriage 68 islowered into the plate cover storage component 72 and the vacuum sourcedisengaged in order to drop the plate cover 44 into the plate coverstorage component 72. Finally, the carriage 68 is then returned to itsoriginal position.

[0149] The plate cover removal station 24 can be adapted to a platecover delivery station 29 by operating the plate cover removal station24 in reverse. The reverse operation causes a plate cover 44 to beremoved from the plate cover storage component 72 and then placed on amultiwell plate 34. When a crystallization system 10 uses both a platecover removal station 24 and a plate cover delivery station 29, theplate covers 44 used with the plate cover removal station 24 can be thesame as or different from the plate covers 44 used with the plate coverdelivery station 29.

[0150]FIG. 4F is a top view of a mother liquor delivery station 26 wherea mother liquor is delivered into the wells 38 of a multiwell plate 34.The mother liquor delivery station 26 includes a plurality of deliveryshuttles 74. Each shuttle includes a delivery block 76 configured toslide along block supports 78. The delivery blocks 76 are coupled withblock actuators 80 to slide the delivery blocks 76 in a lateraldirection relative to the plate track 14 as illustrated by the arrowlabeled A. Suitable block actuators 80 include, but are not limited to,pneumatic pistons, hydraulic pistons and electric motors.

[0151]FIG. 4G provides a top view of a delivery block 76. A plurality oflumens 82 extend through the delivery block 76. The lumens 82 aredivided into a first delivery group 84 and a second delivery group 86. Afluid injector 88. such as a syringe, can be removably positioned ineach of the lumens 82 as illustrated in FIG. 4H. The lumens 82 in eachdelivery group 84. 86 are arranged on the delivery block 76 so eachfluid injector 88 can be concurrently aligned with a different well 38of a multiwell plate 34. Accordingly, the number of lumens 82 in eachdelivery group 84. 86 is preferably equal to the number of wells 38 inthe multiwell plate 34. For instance, when the multiwell plates 34include 48 wells 38, each delivery group 84, 86 preferably includes 48lumens 82.

[0152] Each fluid injector 88 is in fluid communication with a motherliquor source. More than one fluid injector 88 can be in fluidcommunication with a single mother liquor source. However, each fluidinjector 88 is preferably in fluid communication with a different motherliquor source. FIG. 4H illustrates a mother liquor delivery station 26having five first delivery groups 84 and five second delivery groups 86which each include 48 fluid injectors 88. Accordingly, 480 mother liquorsources are required when each fluid injector 88 is in fluidcommunication with a different mother liquor source.

[0153]FIG. 4I is a sideview of a mother liquor source storage bank 90for holding different mother liquor sources 92. The bank 90 includessource holders 94 arranged in five columns and four rows. Each sourceholder 94 can hold a plurality of mother liquor sources 92 and can beslid in and out of the bank 90 to provide easy access to the motherliquor sources 92 being held by a single source holder 94. When eachmultiwell plate 34 has 48 wells 38, each source holder 94 preferablyholds 12 different mother liquor sources 92. Accordingly, each columncontains 48 mother liquor sources 92 which can each be in fluidcommunication with a different fluid injector 88 included in the samedelivery group 84, 86. As a result, each column of mother liquor sources92 can be associated with a single delivery group 84, 86. A motherliquor source bank 90 can be included on each side of the plate track14. The mother liquor sources 92 positioned on one side of the platetrack 14 can be in fluid communication with the delivery groups 84, 86nearest that side of the track while the mother liquor sources 92positioned on the opposing side of the plate track 14 can be in fluidcommunication with the delivery groups 84. 86 on the opposing side ofthe plate track 14.

[0154] During operation of the mother liquor delivery station 26, theplate transport assembly moves a multiwell plate 34 beneath a particularone of the delivery blocks 76. The block actuators 80 can then move thedelivery blocks 76 so the injectors in a particular delivery group 84,86 are aligned with the wells 38 in the multiwell plate 34. Theparticular delivery block 76 and the particular delivery group 84, 86are associated with the mother liquors which the operator desires to bedelivered into the wells 38 of the multiwell plate 34. The motherliquors are then delivered from the mother liquor sources 92 through thefluid injectors 88 and into the wells 38 which are aligned with themother liquors. The delivery of mother liquid into each of the wells 38can occur concurrently and the same volume of mother liquor ispreferably delivered into each of the wells 38.

[0155]FIG. 4J illustrates a syringe pump for delivering mother liquorfrom mother liquor sources 92 into a well 38 of a multiwell plate 34through a fluid injector 88. Mother liquor flows from a mother liquorsource 92 to the fluid injector 88 through a fluid conduit 96. The fluidconduit 96 is in fluid communication with a syringe 98 positionedbetween the mother liquor source 92 and the fluid injector 88. Thevolume within the syringe 100 can be mechanically compressed andexpanded as illustrated by the arrow labeled A. A first pinch bar 102 ispositioned on an input side of the syringe 104 and a second pinch bar106 is positioned on an output side of the syringe 108. The first pinchbar 102 and the second pinch bar 106 are coupled with a rocker bar 110.In FIG. 4J, the rocker bar 110 occupies a first position where the firstpinch bar 102 has pinched the fluid conduit 96 shut on the input side ofside of the syringe while the output side of the syringe 108 remainsunobstructed. The rocker bar 110 can occupy a second position where thesecond pinch bar 106 has pinched the fluid conduit 96 shut on the outputside of the syringe 108 while the output side of the syringe 108 remainsunobstructed. The rocker bar 110 can be automatically moved between thefirst and second positions as illustrated by the arrow labeled B.

[0156] During operation of the syringe pump, the rocker bar 110 occupiesthe first position and the volume within the syringe 100 is expanded bythe amount of mother liquor to be delivered into a well 38 from thefluid injector 88. Because the fluid conduit 96 on the output side ofthe syringe 108 is pinched closed, the expansion of the volume withinthe syringe 100 by a particular amount causes that particular amount tobe withdrawn from the mother liquor source 92. The rocker bar 110 isthen moved to the second position and the volume within the syringe 100compressed by the amount of mother liquor to be delivered into the well38 through the fluid injector 88. Because the fluid conduit 96 on theinput side of the syringe 104 is closed, the compression of the volumewithin the syringe 100 by the particular amount causes that particularamount to flow through the fluid injector 88 and into the associatedwell 38.

[0157] The mother liquor delivery section discussed above is forillustrative purposes only and many variations are possible. Forinstance, a mother liquor delivery station 26 can include more than fivedelivery shuttles 74 or as few as one. Further, each delivery shuttle 74can include more than two delivery groups or a few as one. When adelivery shuttle 74 includes a single delivery group, the blockactuators 80 can be eliminated and the delivery shuttles 74 can bestationary relative to the plate track 14. Additionally, the combinationof the plate track 14 movement and the delivery block 76 movement can beused to position a particular fluid injector 88 over a particular well38 and the mother liquors can be sequentially delivered into the wells38. Accordingly, a particular mother liquor can be delivered into aparticular well 38.

[0158] FIGS. 5A-5E illustrate various stations 12 that may be includedin a drop formation system 32. It is noted that the drop formationsystem 32 illustrated in regard to FIGS. 5A-5E is adapted for a hangingdrop array crystallization. The drop formation system 32 can be readilymodified for a sitting drop array crystallization by causing the motherliquor drops and molecule solution drops to be deposited on a sittingdrop region 42 of a multiwell plate 34, such as the one illustrated inFIG. 3C, as opposed to on a coverslip.

[0159]FIG. 5A is a top view of a drop formation station 28 and FIG. 5Bis a sideview of the drop formation station 28. The drop formationstation 28 includes a wash basin 112 through which a cleansing solutioncan be flowed. Suitable cleansing solutions include, but are not limitedto, water. The drop formation station 28 also includes a moleculesolution storage component 114 having one or more molecule solutionwells 116 for storing solutions containing the molecule to thecrystallized. The molecule solution wells 116 can be capped for storingthe molecule solutions when the drop formation system 32 is not inoperation. The molecule solution storage component 114 can berefrigerated in order to provide cooling to the molecule solution withinthe molecule solution wells 116. For example, when the molecule solutionis a molecule solution, the solution is preferably kept at 3-4° C.whether the drop formation station 28 is or is not in operation. Thedrop formation station 28 also includes syringe pumps 118 and acoverslip storage component 120 for storing coverslips.

[0160] The drop formation station 28 also includes a pipette holder 122configured to move vertically as indicated by the arrow labeled A andlaterally as indicated by the arrows labeled B. The pipette holder's 122lateral range of motion allows the pipette holder 122 to move to avariety of positions including a position over the wash basin 112 and aposition over the coverslip holder 124. The drop formation station 28also includes a coverslip holder 124 configured to be inverted asindicated by the arrow labeled C. The coverslip holder 124 can movevertically as indicated by the arrow labeled D and laterally asindicated by the arrows labeled E. The pipette holder's 122 lateralrange of motion allows the pipette holder 122 to move to a variety ofpositions including a position over the coverslip storage component 120and several positions over the plate track 14. The drop formationstation 28 also includes a molecule delivery pipette 126 which isconfigured to move vertically as indicated by the arrow labeled F,laterally as indicated by the arrow labeled G and longitudinally asindicated by the arrow labeled H. The longitudinal and lateral ranges ofmotion allow the molecule delivery pipette 126 to be moved to a varietyof positions including a position over each molecule solution well and aplurality of positions over the coverslip holder 124.

[0161] The above movements can be achieved by coupling the pipetteholder 122, coverslip holder 124 and the molecule delivery pipette 126to a variety of different actuators. Suitable actuators include, but arenot limited to, pneumatic pistons, hydraulic pistons and a variety ofmotors.

[0162]FIG. 5C is a sideview of a pipette holder 122. The pipette holder122 includes a pipette support frame 128. The pipette support frame 128holds a number of pipettes 130 equal to the number of wells 38 in acolumn of a multiwell plate 34. The pipettes 130 are held at a spacingwhich approximates the spacing between the wells 38 in the column of themultiwell plate 34. This spacing permits each pipette 130 to beconcurrently aligned with a different well 38 in the column.

[0163] Each pipette 130 includes a valve 132 and a conduit 134 extendingfrom the valve 132 to a syringe pump 118. The syringe pump 118 can beused to draw fluid into the pipettes 130 and to drive fluid out of thepipettes 130. The valve 132 is configured to deliver drops of aparticular size from the pipette 130. These drops are delivered from thepipette 130 until a desired total volume is delivered from the pipette130. Suitable valves 132 include, but are not limited to, piezoelectricvalves and solenoid valves which can be configured to deliver drops assmall as 380 pL. This allows production of mother liquor drops as smallas 380 pL. Further reduction in the drop size delivered by thesepipettes 130 may also be possible, would be desired, and is intended tofall within the scope of the present invention.

[0164] The pipette arrangement used for the molecule delivery pipette126 is similar to the pipette arrangement used for the pipettes 130within the pipette holder 122. Accordingly, the molecule deliverypipette 126 also includes a valve 132 and a conduit 134 extending fromthe valve 132 to a syringe pump 118. The molecule delivery pipette 126is able to produce molecule solution drops as small as 380 pL. Furtherreduction in the drop size delivered may also be possible, would bedesired, and is intended to fall within the scope of the presentinvention.

[0165]FIG. 5D is a sideview of a coverslip holder 124. The coverslipholder 124 includes a frame 136 which supports a plurality of supportcups 138 shaped to removably hold coverslips at a spacing whichapproximates the spacing between the wells 38 in a column of themultiwell plate 34. This spacing permits each coverslip to beconcurrently aligned with a different well 38 in a column of themultiwell plate 34.

[0166] The support cups 138 can include an attachment mechanism 140 forimmobilizing the coverslips in place relative to the support cups 138.The attachment mechanism 140 serves to keep the coverslips in place whenthe coverslip holder 124 is inverted. However, the attachment mechanisms140 can release the coverslips at a desired moment. Suitable coverslipholder 124 attachment mechanisms 140 include, but are not limited to, avacuum source in pneumatic communication with vacuum ports positioned inthe support cups 138. Pulling a vacuum through the vacuum ports servesto keep the coverslips in place on the coverslip holder 124. However,when the coverslip holder 124 is inverted, the vacuum can be released bydisengaging the vacuum source or reversing the vacuum. The release ofthe vacuum releases the coverslips from the coverslip holder 124.

[0167]FIG. 5E is a sideview of a coverslip storage component 120 whichincludes a plurality of magazines 142 sized to hold coverslips 144stacked on top of one another. The stack of coverslips 144 within themagazine 142 can be biased upward until the coverslip 144 on the top ofthe stack is near the top of the magazine 142. The spacing between themagazines 142 approximates the spacing between the support cups 138 ofthe coverslip holder 124. This spacing permits each magazine 142 to beconcurrently aligned with a different support cup 138 of the coverslipholder 124. Accordingly, a coverslip 144 from each magazine 142 can alsobe aligned with a different support cup 138.

[0168] FIGS. 6A-61 illustrate a method for operating the drop formationstation 28 to form hanging drops in each of the wells 38 of a multiwellplate 34. The figures are described with respect to crystallization of aprotein, however, the same method can be used for crystallization ofother types of molecules. FIG. 6A illustrates a drop formation station28 in the rest position which can be occupied when the drop formationstation 28 is not in use or between multiwell plates 34 beingtransported into the drop formation station 28. In the rest position,coverslips 144 are attached to the coverslip holder 124 which ispositioned to one side of the plate track 14 and the pipette holder 122is positioned to the opposing side of the plate track 14.

[0169]FIG. 6B illustrates a multiwell plate 34 moved into position fordrop formation and the pipette holder 122 moved into position over thewash basin 112 for priming of the pipettes 130. The pipette holder 122is lowered until the pipette tips are within a cleansing solutionswithin the wash basin 112. Cleansing fluid is aspirated from the washbasin 112 and the pipette holder 122 is raised to remove the pipettetips from the cleansing solution. The cleansing fluid is then expelledfrom the pipettes 130. The process of aspiration and expulsion can berepeated as often as is necessary to achieve a properly primed pipettes130.

[0170]FIG. 6C illustrates the pipette holder 122 moved into positionover a column of wells 38 in the plate 34. The pipette holder 122 ispositioned so each pipette tip is aligned with a different well 38 inthe column. Accordingly, each pipette 130 is associated with aparticular well 38. The pipette holder 122 is lowered until each pipettetip is positioned within the mother liquor in the associated well 38. Aportion of the mother liquor is aspirated from each well 38 associatedwith a pipette tip. The actuators then lift the pipettes 130 upward toremove the pipette tips from the wells 38. A portion of the aspiratedmother liquors are then expelled from each pipette 130. The expelledmother liquors fall back into the associated well 38.

[0171]FIG. 6D illustrates the pipette holder 122 moved over thecoverslip holder 124 and is positioned so each pipette tip is alignedwith a different support cup 138. The support cups 138 are each holdinga coverslip 144 upside down and the attachment mechanism 140 is engagedto immobilize the coverslips 144 relative to the support cups 138. Oneor more drops of mother liquor is expelled from each pipette 130 ontothe associated coverslips 144. As a result, one more drops of the motherliquor from a particular well 38 is delivered onto a particularcoverslip 144.

[0172] The drops of mother liquor are expelled onto the coverslips 144until a desired volume of mother liquor has been delivered onto eachcoverslip 144. The total volume of the drops delivered onto thecoverslips 144 is strictly controlled. As discussed previously, afeature of the present invention is the ability to deliver small volumesprecisely which enables small drop volumes to be used. With deviceswhich can deliver volumes as low as 380 pL, volumes can be deliveredwith great precision. The precision of the volumes delivered ispreferably less than about 25 nL, more preferably less than 20 nL, morepreferably less than 15 nL, and most preferably less than 10 nL. Theprecision of the volumes delivered may also be between 380 pL and 25 nL,more preferably between 380 pL and 20 nL, more preferably between 380 pLand 15 nL, and most preferably between 380 pL and 10 nL.

[0173]FIG. 6E illustrates the pipette holder 122 returned to the restposition which was illustrated in FIG. 6A. The molecule delivery pipette126 is moved into position over a coverslip 144. Before being moved intoposition over the coverslip 144, the molecule delivery pipette 126 waslowered into a particular molecule solution well and a volume of themolecule solution aspirated. Once the molecule delivery pipette 126 isin position over the coverslip 144, drops of the molecule solution aredelivered onto the mother liquor which was previously delivered onto thecoverslip 144. The drops of molecule solution are delivered until adesired volume of molecule solution is achieved on the coverslip 144.The precision of the volumes delivered is preferably less than about 25nL, more preferably less than 20 nL, more preferably less than 15 nL,and most preferably less than 10 nL. The precision of the volumesdelivered may also be between 2 and 25 nL, more preferably between 2 and20 nL, more preferably between 2 and 15 nL, and most preferably between2 and 10 nL.

[0174] The mother liquor drops and the protein drops may be delivered inany order. Once both drops are delivered, the drops combine to form ahanging drop to be studied for crystal formation.

[0175] After forming a hanging drop on the coverslip 144, the moleculedelivery pipette 126 proceeds to the next coverslip 144 until a hangingdrop is formed on each coverslip 144. The molecule delivery pipette 126then returns to the position over the molecule solution well which wasthe source for the molecule solution used to create the hanging drops.The molecule solution remaining in the molecule delivery pipette 126 isexpelled into the molecule solution well.

[0176]FIG. 6F illustrates the molecule delivery pipette 126 returned toits rest position as illustrated in FIG. 6A. FIG. 6F also illustratesthe coverslip holder 124 inverted and moved into position over thecolumn of wells 38 on the multiwell plate 34. The coverslip holder 124is positioned so each coverslip 144 is aligned with a different well 38in the column. Specifically, a given coverslip 144 is aligned with thewell 38 which was the source of the mother liquor used to create thehanging drop on the given well 38.

[0177] The coverslip holder 124 is lowered until the coverslips 144contact the upper edges 40 of the associated wells 38. The sealingmedium which was previously applied to the upper edge 40 of the wells 38causes a seal to be formed between the coverslips 144 and the upperedges 40 of the associated wells 38. The attachment mechanism 140 isreleased and the coverslip holder 124 is raised to leave each coverslip144 in place over an associated well 38. The hanging drop hangs from thecoverslips 144 into the wells 38 as illustrated in FIG. 6G.

[0178]FIG. 6H illustrates the coverslip holder 124 moved into positionover the coverslip storage component 120. The coverslip holder 124 ispositioned so each support cup 138 is aligned with a magazine 142 in thecoverslip storage component 120. Accordingly, each support cup 138 isassociated with the top coverslip 144 in each magazine 142. Thecoverslip holder 124 is lowered until each support cup 138 contacts acoverslip 144 within the associated magazine 142. The attachmentmechanism 140 is engaged to immobilize the contacted coverslips 144relative to the associated support cups 138.

[0179]FIG. 6I illustrates the coverslip holder 124 returned to its restposition. The top coverslip 144 from each magazine 142 discussed withrespect to FIG. 6G is attached to the associated support cup 138.

[0180] The steps described with respect to FIGS. 6A-6I result in ahanging drop being formed in each well 38 of a single column of wells38. These steps are repeated until a hanging drop is formed in the wells38 of each column of the multiwell plate 34. Once a hanging drop isformed in each of the wells 38, the multiwell plate 34 can be moved tothe next station.

[0181] The crystallization system 10 described above can be adapted toform sitting drops. This adaptation can be made with changes to themother liquor delivery station 26 and the drop formation station 28. Forinstance, the mother liquor delivery station 26 is adapted to delivermother liquor into the well regions 41 of a multiwell plate 34 adaptedto perform a sitting drop array microcrystallization such as themultiwell plate 34 illustrated in FIG. 3C. Specifically, the fluidinjectors 88 of the mother liquor delivery station 26 must be alignedwith the well regions 41 before the mother liquor is delivered into thewells 38 of the multiwell plate 34. This alignment permits delivery ofthe mother liquors into the well region 41 of each well 38 withoutdelivering the mother liquors onto the sitting drop region 42 of eachwell 38.

[0182] Adapting the crystallization system 10 to form sitting drops alsoincludes adapting the drop formation station 28 to form sitting drops.The drop formation station 28 can include each of the componentsillustrated in FIG. 5A-5E arranged with the same spatial relationshipsillustrated in FIGS. 5A-5E. However, the method of operating thesecomponents varies from the method illustrated in FIGS. 6A-6I. FIGS.7A-7G illustrate a method for operating the drop formation station 28 toform sitting drops in each well 38 of a multiwell plate 34 adapted toperform a sitting drop array microcrystallization.

[0183] The figures are described with respect to crystallization of aprotein, however, the same method can be used for crystallization ofother types of molecules. FIG. 7A illustrates the drop formation station28 in the same rest position illustrated in FIG. 6A. FIG. 7B illustratesa multiwell plate 34 adapted to perform a sitting drop arraymicrocrystallization moved into position for sitting drop formation.Accordingly, each well 38 in the multiwell plate 34 includes a wellregion 41 adjacent to a sitting drop region 42. FIG. 7B also illustratesthe pipette holder 122 moved into position over the wash basin 112 forpriming of the pipettes. The pipettes are primed as described withrespect to FIG. 6B.

[0184]FIG. 7C illustrates the pipette holder 122 moved into positionover a column of wells 38 in the multiwell plate 34. The pipette holder122 is positioned so each pipette tip is aligned with the well region 41in a different well 38 in the column as illustrated in FIG. 7D.Accordingly, each pipette is associated with a particular well 38. Thepipette holder 122 is lowered until the tip of each pipette ispositioned in the mother liquor which was previously delivered into thewell region 41 of the associated well 38. A portion of the mother liquoris aspirated from each well region 41 associated with a pipette tip. Theactuators then lift the pipette upward to remove the pipette tips fromthe wells 38. A portion of the aspirated mother liquors are thenexpelled from each pipette. The expelled mother liquors fall back intothe associated well regions 41.

[0185] The pipette holder 122 is then moved so each pipette tip isaligned with the sitting drop region 42 in a different well 38 in thecolumn as illustrated in FIG. 7E. One or more drops of mother liquor isexpelled from each pipette onto the associated sitting drop region 42.As a result, one more drops of the mother liquor from a particular wellregion 41 is delivered onto the sitting drop region 42 of the same well38. The drops of mother liquor are expelled onto the sitting drop region42 until a desired volume of mother liquor has been delivered onto eachsitting drop region 42. The total volume of the drops delivered onto thecoverslips 144 is strictly controlled. As discussed previously, afeature of the present invention is the ability to deliver small volumesprecisely which enables small drop volumes to be used. With deviceswhich can deliver volumes as low as 380 pL, volumes can be deliveredwith great precision. The precision of the volumes delivered ispreferably less than about 25 nL, more preferably less than 20 nL, morepreferably less than 15 nL, and most preferably less than 10 nL.

[0186] The precision of the volumes delivered may also be between 380 pLand 25 nL, more preferably between 386 pL and 20 nL, more preferablybetween 380 pL and 15 nL, and most preferably between 380 pL and 10 nL.

[0187]FIG. 7F illustrates the pipette holder 122 returned to the restposition which was illustrated in FIG. 7A. The molecule delivery pipette126 is moved into position over a sitting drop region 42 in a well 38 ofthe column. Before being moved into position over the well 38, themolecule delivery pipette 126 was lowered into a particular moleculesolution well and a volume of the molecule solution aspirated. Once themolecule delivery pipette 126 is in position over the sitting dropregion 42, drops of the molecule solution are delivered onto the motherliquor which was previously delivered onto the coverslip 144. The dropsof molecule solution are delivered until a desired volume of moleculesolution is achieved on the sitting drop region 42. The precision of thevolumes delivered is preferably less than about 25 nL, more preferablyless than 20 nL, more preferably less than 15 nL, and most preferablyless than 10 nL. The precision of the volumes delivered may also bebetween 380 pL and 25 nL, more preferably between 380 pL and 20 nL, morepreferably between 380 pL and 15 nL, and most preferably between 380 pLand 10 nL.

[0188] The mother liquor drops and the protein drops may be delivered inany order. Once both drops are delivered, the drops combine to form asitting drop to be studied for crystal formation. FIG. 7G illustrates asitting drops formed on the sitting drop region 42 of a well 38. Afterforming the sitting drop on the sitting drop region 42, the moleculedelivery pipette 126 proceeds to the sitting drop region 42 in the nextwell 38 until a sitting drop is formed in each well 38 of the column.The molecule delivery pipette 126 then returns to the position over themolecule solution well which was the source for the molecule solutionused to create the sitting drops. The molecule solution remaining in themolecule delivery pipette 126 is expelled into the molecule solutionwell.

[0189] After formation of the sitting drop, the coverslips 144 arepositioned over the wells 38 having sitting drops, new cover slips areloaded onto the converslip holder 124 and the drop formation station 28is returned to the rest position as described above with respect to FIG.6F-6I.

[0190] The steps described with respect to FIGS. 7A-7G result in asitting drop being formed in each well 38 of a single column of wells38. These steps are repeated until a hanging drop is formed in the wells38 of each column of the multiwell plate 34. Once a hanging drop isformed in each of the wells 38, the multiwell plate 34 can be moved tothe next station.

[0191] Although FIGS. 6A-7G illustrate a method for operating the dropformation station to form sitting drops and hanging drops, the hangingdrop station be easily adapted to other crystallization techniques,other well geometries and/or other multiwell plate geometries.

[0192] It is noted that the apparatuses described in regard to FIGS.6A-7G may optionally include one or more sensors which can detectwhether mother liquor drops and/or molecule drops have been formed. Anexample of a suitable sensor is a LED sensor.

[0193] While many plate tracks 14 and transport assemblies can be usedwith the above stations 12, FIGS. 8A-8C illustrate a preferredembodiment of a plate track 14 for transporting multiwell plates 34between the above stations 12. FIGS. 8A and 8B are sideviews of a platetrack 14 looking down the longitudinal axis of the plate track 14. Theplate track 14 includes two spaced apart plate supports 158. A pin 160extends upward from a pin carriage 162 positioned beneath the platetrack 14. The carriage includes mechanics which can be actuated toextend the pin 160 above the plate track 14 as illustrated in FIG. 8A orto withdraw the pin 160 below the plate track 14 as illustrated in FIG.8B.

[0194]FIG. 8C is a lateral sideview of a plate track 14 and transportassembly with a plurality of multiwell plates 34 present on the platetrack 14. The transport assembly includes a first pin carriage 162A, asecond pin carriage 162B and a third pin carriage 162C. Each of the pincarriages 162A, 162B, 162C is configured to move along the longitudinalaxis of the plate track 14 as illustrated by the arrows labeled A, B andC. The brackets at the ends of the arrows indicate the range of motionof each pin carriage 162A, 162B, 162C.

[0195] The first pin carriage 162A and the third pin carriage 162Cinclude a plurality of pins 160. The pins are located along the pincarriage 162A, 162C with an approximately constant displacement betweenadjacent pins 160. The pin carriage 162A, 162C serves to maintain thedisplacement between the pins 160 during movement of the pin carriage162A, 162C.

[0196] Each pin 160 is illustrated in the extended position, however,the pins in one pin carriage 162 can be withdrawn while the pins 160 inanother pin carriage 162 are extended. In another embodiment, a portionof the pins 160 in a single pin carriage 162 can be extended whileanother portion of the pins 160 within the same pin carriage 162 arewithdrawn.

[0197] An air gap 166 is formed between the pin carriages 162A, 162B,162C and each of the multiwell plates 34 positioned on the plate track14 so the pin carriages 162A, 162B, 162C do not contact the bottomsurface of the multiwell plates 34. As a result, when the pins 160 arewithdrawn, the pin carriages 162A, 162B, 162C can be moved along thelongitudinal axis of the plate track 14 without moving multiwell plate34 on the plate track 14. When the pin 160 is extended and the pincarriage 162 is moved along the longitudinal axis of the plate track 14,the pin 160 pushes any multiwell plate 34 obstructing the pin's travelalong the longitudinal axis of the plate track 14.

[0198] As described above, the plate transport assembly is used totransport the multiwell plates 34 from station to station along theplate track 14. Various positions along the plate track 14 can beassociated with a particular station of the crystallization system 10.For instance, when a multiwell plate 34 is located at position P₁, a barcode on the multiwell plate 34 can be read by a bar code station andwhen a multiwell plate 34 is located at position P₂, a sealing mediumstation 22 can be used to apply a sealing medium to the upper edge 40 ofthe wells 38 of the multiwell plate 34. Further, when a multiwell plate34 is positioned at P₃, the multiwell plate 34 can be positioned beneathone of the delivery shuttles 74 of a mother liquor delivery station 26.

[0199] The following description describes a method for using the abovetransport assembly for advancing the multiwell plate 34 labeled T₁ fromthe position labeled P₁ to the position labeled P₂ to the positionlabeled P₃. Once the multiwell plate T₁ is located at position P₁, thepins 160 in the first pin carriage 162A are withdrawn below the platetrack 14. The first pin carriage 162A is then moved to the left and thepins 160 extended above the plate track 14 as illustrated in FIG. 8C.The multiwell plate T₁ can then be moved from position P₁ to position P₂by moving the first pin carriage 162A to the right until the multiwellplate T₁ is positioned at position P₂. The pins 160 are then withdrawnbelow the plate track 14 and the first pin carriage 162A is moved backto is original position and the pins 160 are again extended above theplate track 14. The pins 160 in the second pin carriage 162B arewithdrawn below the plate track 14 and the second pin carriage 162B ismoved to the left of the multiwell plate T₁. The pins 160 in the secondpin carriage 162B are then extended above the plate track 14 and thesecond pin carriage 162B moved to the right until the multiwell plate T₁is located at position P₃.

[0200] The plurality of pin carriages 162 illustrated in FIG. 8C allowsa multiwell plate 34 at one station to be processed through thecrystallization system 10 independently of another multiwell plate 34being processed through the crystallization system 10. For instance, afirst multiwell plate 34 can be advanced from P₁ to P₂ while a secondmultiwell plate 34 remains in place at P₃. As a result, when P₁, P₂ andP₃ are each associated with different stations 12, multiwell plates 34can be processed through different stations 12 at different rates.Further, different pin carriages 162 which make up a transport assemblycan be included with independent systems which are assembled together toform the system. For instance, the first pin carriage 162A and thesecond pin carriage 162B can be included in a mother liquor deliversystem and the third pin carriage 162C can be included in a dropformation system 32.

[0201] Crystal formation can be detected by examining each drop for theformation of crystals. In a preferred embodiment, crystals are detectedand graded in the various wells for crystal quality. This may be donemanually or by an automated device. Diversified Scientific, Inc. ofBirmingham, Ala. manufactures CRYSTALSCORE™ which may be used toautomate the scoring of crystal formation.

[0202] As described above, the system can be used to performedcrystallization trials where various mother liquor are screened fortheir ability to crystallize a protein of interest. The crystallizationtrials frequently include a coarse screen followed by one or more finescreens. While the mother liquors used for the fine screens are oftendependent on the results of the coarse screen, the mother liquors usedfor the coarse screen can be standard for each crystallization trial.

[0203] When the mother liquors are used to crystallize proteins, apreferred coarse screen preferably consists of the 15 sub-screens listedin Table 1. The number of mother liquors included in each sub-screen isalso listed in Table 1. The composition of the mother liquors includedeach of these sub-screens is listed in FIG. 9. Mother liquors having thelisted compositions can be obtained from Hampton Research of LagunaNiguel, Calif.

[0204] As illustrated in Table 1, a total of 480 mother liquors areassociated with the sub-screens of the preferred coarse screen. Since480 mother liquors are included in the coarse screen and since eachplate preferably includes 48 wells, the coarse screen can be performedby processing only 10 plates through the system. Further, thesub-screens generally include 24 or 48 mother liquors. Accordingly, eachplate can include from one to two sub-screens. TABLE 1 Screen Number ofmother liquors Crystal screen I 48 Crystal screen II 48 Grid ammoniumsulfate 24 Grid MPD 24 Grid sodium chloride 24 Grid PEG6000 24 GridPEG/lithium chloride 24 Sodium/potassium phosphate 24 PEG/ion screen 48Membrane protein screen 48 Detergent screen I 24 Detergent screen II 24Detergent screen III 24 Cryo screen 48 Low ionic strength screen 24

[0205] Each of the mother liquors used for the coarse screen can bestored in one or more of the mother liquor storage banks. However, thenumber of mother liquors which may be needed for different fine screensis large enough that storage of these mother liquors impractical.Accordingly, the system can also include a station which forms the finescreen mother liquors from stock solutions and then delivers them intothe wells of a plate. Alternatively, one or more external systems can beused to create the fine screen mother liquors from stock solutions andto deliver these mother liquors into the wells of one or more plates.These plates can then be processed through the system.

[0206] When an external system is used to form and deliver fine screenmother liquors, the system control logic needs to override the motherliquor delivery station in order to avoid doubling up on the delivery ofmother liquor into the wells of a plate. As a result, the system controllogic must be informed when a plate which already has mother liquor isin the system. An operator can use a user interface to inform the systemcontrol logic which one of the plates already has mother liquorsdelivered into the wells. Alternatively, an operator use a plate havinga bar code which indicates that mother liquors are already present inthe wells of the plate.

EXAMPLE 1

[0207] The system described above was used in a plurality of lysozymecrystallization experiments where lysozyme was crystallized in a motherliquor composition including 100 mM sodium acetate and 10% sodiumchloride at a pH of 4.6. The volume of the hanging drop formed by thedrop formation station was different for each experiment. FIGS. 10A-10Drespectively illustrate crystal formed in hanging drops of 40 nL, 100nL, 200 nL and 1000 nL. The crystals were formed regardless of thereduction in drop size. As a result, the system can be used withsubmicroliter hanging drop volumes.

EXAMPLE 2

[0208] The system described above was used in a crystallization trialwhere the mother liquor for crystallizing lysozyme was optimized. Duringthe coarse screen, 480 crystallization experiments were performed usingeach of the 480 mother liquors disclosed in FIG. 9. The results fromeach of the 480 experiments were compared to one another to identify oneor more crystallization experiments yielding crystals with the mostdesirable characteristics. One of the identified coarse screenexperiments was associated with a mother liquor composed of 30% MPD(+/−2-methyl-2,4-pentanediol), 100 mM sodium acetate, 20 mM calciumchloride, at pH 4.6.

[0209] A fine screen consisting of 24 crystallization experiments wasthen performed. The composition of the mother liquors associated witheach of the 24 crystallization experiments was selected relative to thecomposition of the mother liquor associated with the identified coarsescreen experiment. The compositions of the 24 mother liquors selectedfor the crystallization experiments of the fine screen are listed inFIG. 11. The concentrations of certain components in each of the 24mother liquors matched the concentration of these components in theidentified coarse screen experiment. For instance, the mother liquorassociated with the identified coarse screen experiment and the motherliquors for each of the fine screen crystallization experiments were allabout 30% MPD and 100 mM sodium acetate. The concentrations of othercomponents in the 24 mother liquors were varied over a range whichencompassed the concentration of these same components in the identifiedcoarse screen experiment. For instance, the concentration of calciumchloride was 20 mM in the identified coarse screen experiment but wasvaried from 12.5-27.5 mM in the 24 mother liquors. Similarly, the pH was4.6 in the identified coarse screen crystallization experiment but wasvaried from 4.1 to 5.1 in the 24 mother liquors.

[0210] Each of the 24 fine screen crystallization experiments werecompared to one another to identify the one or more crystallizationexperiments yielding the most desirable characteristics.

[0211] The foregoing examples and description of preferred embodimentsof the present invention are provided for the purposes of illustrationand description. The examples and preferred embodiments, however, arenot intended to be exhaustive or to limit the invention to the preciseforms disclosed. Obviously, many modifications and variations will beapparent. to practitioners skilled in this art. The embodiments werechosen and described in order to best explain the principles of theinvention and its practical application, thereby enabling others skilledin the art to understand the invention for various embodiments and withvarious modifications as are suited to the particular use contemplated.It is intended that the scope of the invention be defined by thefollowing claims and their equivalents.

What is claimed is:
 1. A method for performing arraymicrocrystallizations to determine suitable crystallization conditionsfor a molecule, the method comprising: forming an array ofmicrocrystallizations, each microcrystallization comprising a dropcomprising a mother liquor solution whose composition varies within thearray and a molecule to be crystallized, the drop having a volume ofless than 1 microliter; storing the array of microcrystallizations underconditions suitable for molecule crystals to form in the drops in thearray; and detecting molecule crystal formation in the drops by takingimages of the drops.
 2. The method according to claim 1 wherein takingimages of the drops comprises taking a single image of each drop.
 3. Themethod according to claim 1 wherein taking images of the drops isperformed without having to adjust a focus of an optical system takingthe images.
 4. The method according to claim 3 wherein taking images ofthe drops comprises taking a single image of each drop.
 5. The methodaccording to claim 1 wherein the molecule is a macromolecule.
 6. Themethod according to claim 1 wherein the molecule is a protein.
 7. Themethod according to claim 1 wherein the macromolecule has a molecularweight of at least 500 daltons.
 8. The method according to claim 1wherein the drop has a volume of less than about 750 nL.
 9. The methodaccording to claim 1 wherein the drop has a volume of less than about500 nL.
 10. The method according to claim 1 wherein the drop has avolume of less than about 250 nL.
 11. The method according to claim 1wherein the drop has a volume of between about 1 nL-750 nL.
 12. Themethod according to claim 1 wherein the drop has a volume of betweenabout 1 nL-500 nL.
 13. The method according to claim 1 wherein the drophas a volume of between about 1 nL-250 nL.
 14. The method according toclaim 1 wherein each microcrystallization further includes a volume ofmother liquor solution separate from the drop, the mother liquorsolution contained in the volume having the same composition as themother liquor solution contained in the drop, the volume comprising lessthan about 500 mL of the mother liquor solution.
 15. The methodaccording to claim 1 wherein each microcrystallization further includesa volume of mother liquor solution separate from the drop, the motherliquor solution contained in the volume having the same composition asthe mother liquor solution contained in the drop, the volume comprisingless than about 250 mL of the mother liquor solution.
 16. The methodaccording to claim 1 wherein the mother liquor solutions have at least 4components which are varied within the array.
 17. The method accordingto claim 1 wherein the mother liquor solutions have at least 5components which are varied within the array.
 18. The method accordingto claim 1 wherein the array includes greater than 96microcrystallizations.
 19. The method according to claim 1 wherein thearray includes greater than 192 microcrystallizations.
 20. The methodaccording to claim 1 wherein forming the array of microcrystallizationsincludes using greater than 48 stock solutions to form the mother liquorsolutions used in the array.
 21. The method according to claim 1 whereinforming the array of microcrystallizations includes using greater than96 stock solutions to form the mother liquor solutions used in thearray.
 22. The method according to claim 1 wherein forming the array ofmicrocrystallizations includes using greater than 192 stock solutions toform the mother liquor solutions used in the array.
 23. The methodaccording to claim 1 wherein forming the array of microcrystallizationsincludes forming the drops within a volume range of less than about 25nL.
 24. The method according to claim 1 wherein forming the array ofmicrocrystallizations includes forming the drops within a volume rangeof less than about 20 nL.
 25. The method according to claim 1 whereinforming the array of microcrystallizations includes forming the dropswithin a volume range of less than about 15 nL.
 26. A method forperforming array microcrystallizations to determine suitablecrystallization conditions for a molecule, the method comprising:forming an array of microcrystallizations, each microcrystallizationcomprising a hanging drop comprising a mother liquor solution whosecomposition varies within the array and a molecule to be crystallized,the drop having a volume of less than 1 microliter; storing the array ofmicrocrystallizations under conditions suitable for molecule crystals toform in the drops in the array; and detecting molecule crystal formationin the drops by taking images of the drops.
 27. The method according toclaim 26 wherein taking images of the drops comprises taking a singleimage of each drop.
 28. The method according to claim 26 wherein takingimages of the drops is performed without having to adjust a focus of anoptical system taking the images.
 29. The method according to claim 28wherein taking images of the drops comprises taking a single image ofeach drop.
 30. A method for performing array microcrystallizations todetermine suitable crystallization conditions for a molecule, the methodcomprising: forming an array of microcrystallizations, eachmicrocrystallization comprising a sitting drop comprising a motherliquor solution whose composition varies within the array and a moleculeto be crystallized, the drop having a volume of less than 1 microliter;storing the array of microcrystallizations under conditions suitable formolecule crystals to form in the drops in the array; and detectingmolecule crystal formation in the drops by taking images of the drops.31. The method according to claim 30 wherein taking images of the dropscomprises taking a single image of each drop.
 32. The method accordingto claim 30 wherein taking images of the drops is performed withouthaving to adjust a focus of an optical system taking the images.
 33. Themethod according to claim 32 wherein taking images of the dropscomprises taking a single image of each drop.
 34. A method forperforming array microcrystallizations to determine suitablecrystallization conditions for a molecule, the method comprising:forming an array of microcrystallizations, each microcrystallizationcomprising a microcrystallization volume comprising a mother liquorsolution whose composition varies within the array and a molecule to becrystallized, the microcrystallization volume having a volume of lessthan 1 microliter; storing the array of microcrystallizations underconditions suitable for molecule crystals to form in themicrocrystallization volumes; and detecting molecule crystal formationin the microcrystallization volumes by taking images of themicrocrystallization volumes.
 35. The method according to claim 34wherein taking images of the microcrystallization volumes comprisestaking a single image of each microcrystallization volume.
 36. Themethod according to claim 34 wherein taking images of themicrocrystallization volumes is performed without having to adjust afocus of an optical system taking the images.
 37. The method accordingto claim 36 wherein taking images of the microcrystallization volumescomprises taking a single image of each microcrystallization volume. 38.The method according to claim 37 wherein the molecule is amacromolecule.
 39. The method according to claim 37 wherein the moleculeis a protein.
 40. The method according to claim 37 wherein themacromolecule has a molecular weight of at least 500 daltons.
 41. Themethod according to claim 37 wherein the microcrystallization volume hasa volume of less than about 750 nL.
 42. The method according to claim 37wherein the microcrystalization volume has a volume of less than about500 nL.
 43. The method according to claim 37 wherein themicrocrystalization volume has a volume of less than about 250 nL. 44.The method according to claim 37 wherein the microcrystalization volumehas a volume of between about 1 nL-750 nL.
 45. The method according toclaim 37 wherein the microcrystalization volume has a volume of betweenabout 1 nL-500 nL.
 46. The method according to claim 37 wherein themicrocrystalization volume has a volume of between about 1 nL-250 nL.47. The method according to claim 37 wherein the mother liquor solutionshave at least 4 components which are varied within the array.
 48. Themethod according to claim 37 wherein the mother liquor solutions have atleast 5 components which are varied within the array.
 49. The methodaccording to claim 37 wherein the array includes greater than 96microcrystallizations.
 50. The method according to claim 37 wherein thearray includes greater than 192 microcrystallizations.
 51. The methodaccording to claim 37 wherein forming the array of microcrystallizationsincludes using greater than 48 stock solutions to form the mother liquorsolutions used in the array.
 52. The method according to claim 37wherein forming the array of microcrystallizations includes usinggreater than 96 stock solutions to form the mother liquor solutions usedin the array.
 53. The method according to claim 37 wherein forming thearray of microcrystallizations includes using greater than 192 stocksolutions to form the mother liquor solutions used in the array.
 54. Themethod according to claim 37 wherein forming the array ofmicrocrystallizations includes forming the microcrystallization volumeswithin a volume range of less than about 25 nL.
 55. The method accordingto claim 37 wherein forming the array of microcrystallizations includesforming the microcrystallization volumes within a volume range of lessthan about 20 nL.
 56. The method according to claim 37 wherein formingthe array of microcrystallizations includes forming themicrocrystallization volumes within a volume range of less than about 15nL.